Project Summary The slow afterhyperpolarization (sAHP) is a calcium-activated potassium conductance that follows a surge of neuronal activity, controlling the number of action potentials fired, and consequently neuronal excitability. Importantly, the sAHP is the major potassium conductance targeted by almost all cortical neuromodulators and neurotransmitters that activate the second messenger cAMP and its main downstream effector protein kinase A (PKA); this includes norepinephrine (NE), serotonin, dopamine, and vasoactive intestinal peptide (VIP). As a result, changes to the sAHP correlate with several cognitive functions, including acquisition or retrieval of memories. Although progress over the last ten years has been made to identify the molecular components of the sAHP, the identity of the potassium channels that open during the sAHP, and how these channels are modulated by PKA are still not clear. This proposal seeks to use a new alternative to traditional cloning approaches to link potassium channels to the sAHP. Our strategy is highly innovative as it combines both a computational and experimental approach. First, we make use of the recent publication of the single cell exome from mouse cortex to identify candidate potassium channels present in sAHP-containing cortical areas. Then, to further filter these candidates, we developed a new strategy to predict PKA substrates on potassium channels, which is loosely based on our previously developed scan-x program. The new platform boasts a variety of unique improvements including probability-based scoring and the treatment of multiple PKA motifs independently in predictor generation. Experimentally, we will use the CRISPR/Cas9 genome editing system to test which potassium channels contribute to the sAHP. This state-of-the-art methodology allows us to compare and contrast the effects of multiple genes simultaneously and in a short time frame using in utero electroporation (IUE), which would not be possible with a standard transgenic mouse approach.